Inhibition of the protective effects of preconditioning in ischemia–reperfusion injury by chronic methadone: the role of pAkt and pSTAT3

Cardiac ischemic preconditioning (Pre) reduces cardiac ischemia–reperfusion injury (IRI) by stimulating opioid receptors. Chronic use of opioids can alter the signaling pathways. We investigated the effects of chronic methadone use on IRI and Pre. The experiments were performed on isolated hearts of male Wistar rats in four groups: IRI, Methadone + IRI (M-IRI), Pre + IRI (Pre-IRI), Methadone + Pre + IRI (M-Pre-IRI). The infarct size (IS) in the Pre-IRI group was smaller than the IRI group (26.8% vs. 47.8%, P < 0.05). In the M-IRI and M-Pre-IRI groups, the infarct size was similar to the IRI group. Akt (Ak strain transforming) phosphorylation in the Pre-IRI, M-IRI, and M-Pre-IRI groups was significantly higher than in the IRI group (0.56 ± 0.15, 0.63 ± 0.20, and 0.93 ± 0.18 vs 0.28 ± 0.17 respectively). STAT3 (signal transducer and activator of transcription 3) phosphorylation in the Pre-IRI and M-Pre-IRI groups (1.38 ± 0.14 and 1.46 ± 0.33) was significantly higher than the IRI and M-IRI groups (0.99 ± 0.1 and 0.98 ± 0.2). Thus, chronic use of methadone not only has no protective effect against IRI but also destroys the protective effects of ischemic preconditioning. This may be due to the hyperactivation of Akt and changes in signaling pathways.

and reporting.After a week of adaptation, the animals were randomly placed in four groups of twelve (7 hearts for measuring functional heart indices and chemical and molecular investigations and 5 hearts for staining and assessment of the size of the infarcted area) and they received drinking water or drinking water containing methadone for four weeks.At the age of thirteen weeks, experiments were performed on the isolated hearts.
1. Ischemia-reperfusion injury (IRI) group: after receiving 4 weeks of normal water as methadone solvent, the rats' hearts were transferred to the Langendorff system and exposed to ischemia-reperfusion. 2. Methadone + IRI (M-IRI) group: After receiving 4 weeks of increasing doses of methadone, the rats' hearts were transferred to the Langendorff system and exposed to ischemia-reperfusion. 3. Pre + IRI (Pre-IRI) group: After receiving 4 weeks of water as methadone solvent, the rats' hearts were transferred to the Langendorff system, and before long-term global ischemia-reperfusion induction, the hearts were exposed to global ischemia for three periods of five minutes, all of the ischemia periods were followed by five minutes of reperfusion.4. Methadone + Pre + IRI (M-Pre-IRI) group: In this group, the animals were exposed to incremental doses of methadone for four weeks, similar to the second group, and at the end of the fourth week, the hearts of these animals were exposed to ischemic preconditioning and ischemia-reperfusion as in the third group.

Chronic use of methadone
Methadone was added to the animals' drinking water incrementally.On the first two days 0.1 mg/ml, on days three and four 0.2 mg/ml, on days five and six 0.3 mg/ml, and from day seven to the end of the study 0.4 mg/ml of methadone was added to the drinking water 30 .

Inducing ischemia-reperfusion injury and preconditioning
Ischemia-reperfusion injury and preconditioning were induced on the isolated heart in the Langendorff system.
To prevent blood clots in the animal, first, 500 units of heparin per 100 g of weight were injected intraperitoneally.Ten minutes after the heparin injection, the rats were anesthetized using sodium thiopental at the dose of 50 mg per kilogram of body weight 31 .Then, by making a slit in the abdomen that extended to both sides of the chest, the rat's heart was quickly removed and transferred to the Langendorff system.The heart was retrogradely perfused with Krebs solution (pH 7.4) at a temperature of 37 °C (containing glucose 11, NaCl 118 mmol/L, KCL 4.8 mmol/L, MgSO 4 1.2 mmol/L, KH 2 PO 4 1.2 mmol/L, NaHCO 3 25 mmol/L, and CaCl 2 1.2 mmol/L), which was aerated with carbogen gas mixture (95% oxygen and 5% carbon dioxide), through the aorta.The perfusion pressure was kept constant at 70 mmHg during the experiment.Left ventricular function was monitored through a ballon, which was connected to a pressure transducer and sent into the left ventricle through the left atrium.Before starting the experiments, the hearts were given 20 min to adapt to the conditions (stabilization phase).Then, to induce ischemia-reperfusion, the hearts were exposed to ischemia for 30 min, followed by reperfusion for 60 min.To create global ischemia, the Krebs solution flow into the aorta was cut off for 30 min and re-established to create reperfusion.In the preconditioning groups, immediately before the start of ischemia, the hearts were exposed to three periods of short-term ischemia-reperfusion (five minutes of ischemia, five minutes of reperfusion) 14 .During all the mentioned stages, the rate of left ventricular developed pressure (LVDevP), left ventricular end-diastolic pressure (LVEDP), and + dP/dtmax was recorded using the Power lab physiograph system.

Heart damage determination
The degree of heart damage was assessed by measuring the level of cardiac troponin I in the coronary effluent at the end of the experiment.Triphenyl tetrazolium chloride (TTC) staining was used to measure the infarcted area of the heart.At the end of the experiments, the heart was removed from the device and 3-mm slices were prepared on the transverse axis.The slices were incubated for 15 min in 1.5% TTC solution in phosphate buffer at 37 °C.Then, the stained sections were placed in 4% paraformaldehyde for 48 h.Finally, using ImageJ software (https:// imagej.net/ ij/ downl oad.html), the surface area of the infarcted area was determined and expressed as a percentage of the total area of the heart.The cardiac troponin I level was measured using a cardiac troponin I ELISA kit (ab24652) manufactured by Abcam (United States).

Measurement of Akt and STAT3 phosphorylation using the western blot method
Heart tissue samples were homogenized on ice in a cold RIPA buffer.The homogenate was centrifuged at 4 °C for 20 min at 14,000×g.The supernatant solution was separated, and its protein concentration was determined using the BCA Protein Quantification kit and according to the manufacturer's instructions.An equal volume of 2 × Laemmli sample buffer was added to the lysate (containing 15 μg of protein) and boiled for 5 min.

Statistical analysis
Data were expressed as mean ± SD.All statistical analyses were done by SPSS 26 and GraphPad Prism10.After verifying the normal distribution of data using the Shapiro-Wilk test, one-way ANOVA was used to compare cardiac troponin I and infarct size.Hemodynamic data were analyzed using two-way repeated measure ANOVA.
In the case of significance, pairwise comparisons were conducted by Tukey's post hoc test.P-values < 0.05 were considered to be statistically significant.

Results
As expected, preconditioning significantly reduced the size of the infarcted area compared to ischemia-reperfusion (26.8% ± 8 vs. 47.8% ± 11.5, P < 0.05).Chronic use of methadone had no significant effect on the size of the infarcted area following IRI.However, the beneficial effects of preconditioning were lost.The size of the infarcted area in the M-IRI and M-Pre-IRI groups was 52.4% ± 9.8 and 57.4% ± 10.4 (P < 0.01 and P < 0.001 compared to Pre-IRI), respectively (Fig. 1a,b).
The cardiac troponin I levels in the cardiac effluent of the Pre-IRI group were significantly lower than the IRI group (0.81 ± 0.26 vs. 2.16 ± 0.53 ng/ml, P < 0.001).However, in the M-IRI and M-Pre-IRI groups (2.01 ± 0.68 and 2.13 ± 0.57 ng/ml, respectively) they were similar to the IRI group but significantly more than the Pre-IRI group (P < 0.01) (Fig. 1c).
+ dP/dtmax was the same in all groups at baseline.This index decreased significantly at the end of 60 min of reperfusion in all groups (P < 0.001).At the end of 60 min of reperfusion, dP/dtmax (contractility index) of the left ventricle of the Pre-IRI group was significantly higher than the IRI group (1831.9± 342 vs. 1254.9± 347.8 mmHg/s, P < 0.05).In the M-IRI and M-Pre-IRI groups, + dP/dtmax was similar to the IRI group and significantly lower than the Pre-IRI group (1158.5 ± 308.9 and 1143.4 ± 396.1 mmHg/s, respectively, P < 0.01) (Fig. 2a).
Left ventricular developed pressure was the same in all groups at baseline.However, at the end of 60 min of reperfusion, there was a significant decrease in all groups (P < 0.001).At the end of 60 min of reperfusion, the developed left ventricular pressure of the Pre-IRI group was significantly higher than the IRI group (53.9 ± 6.9 vs. 30.9± 8.3 mmHg, P < 0.001).In the M-IRI and M-Pre-IRI groups, it was similar to the IRI group and significantly lower than the Pre-IRI group (31.3 ± 6.9 and 30.8 ± 8.4 mmHg, respectively, P < 0.001) (Fig. 2b).
Left ventricular end-diastolic pressure was the same in all groups at baseline.However, at the end of 60 min of reperfusion, there was a significant increase in all groups (P < 0.001).The end-diastolic pressure in the Pre-IRI group was significantly lower than the IR group (28.4 ± 7.1 vs. 40.7 ± 7.4 mmHg, P < 0.05) and in the M-IRI and M-Pre-IR groups, it was similar to the IRI group and significantly higher than the Pre-IRI group (42.3 ± 6.5 and 43.1 ± 9.2 mmHg, respectively, P < 0.01) (Fig. 2c).
The level of Akt phosphorylation in the Pre-IRI, M-IRI, and M-Pre-IRI groups was 0.56 ± 0.15, 0.63 ± 0.2, and 0.93 ± 0.18, respectively, which was significantly higher than the IRI group (0.28 ± 0.17) (P < 0.05, P < 0.01, and P < 0.001, respectively).In addition, the level of Akt phosphorylation in the M-Pre-IRI group was significantly higher than in the Pre-IRI and M-IRI groups (P < 0.01 and P < 0.05, respectively) (Fig. 3a and Supplementary File).
The level of STAT3 phosphorylation in the Pre-IRI group was 1.38 ± 0.14, which was significantly higher than the IRI group (P < 0.05, 0.99 ± 0.1), but in the M-IRI group (0.98 ± 0.2) it was similar to the IRI group.STAT3 phosphorylation in the M-Pre-IRI group (1.21 ± 0.3) was significantly higher than the IRI and M-IRI groups (P < 0.01 and P < 0.001, respectively) (Fig. 3b and Supplementary File).

Discussion
The results of this study showed that chronic use of methadone for four weeks in rats had no effect against ischemia-reperfusion.In addition, chronic use of methadone inhibited the protective effects of preconditioning on ischemia-reperfusion injury in the heart.
During ischemic conditioning various factors such as adenosine, bradykinin, acetylcholine, opioids, TNF cytokines play a role.however, the opioid system is one of the most important systems in creating protective effects of ischemic preconditioning 33,34 .In addition, similar to ischemic preconditioning, acute administration of morphine five minutes before inducing ischemia-reperfusion injury reduces the infarct size and prevents arrhythmia after ischemia-reperfusion 12,13,15 .
However, in this study, we showed that chronic use of methadone not only did not have a protective effect against IRI but also blocked the protective effects caused by ischemic preconditioning.In line with our study, Cohen et al. 35 observed in a study that in conscious ischemic rabbits, preconditioning, if applied for a short period, has a protective effect against ischemia-reperfusion, but if the ischemic preconditioning cycles are increased to about 40 to 65 cycles during 3 to 4 days before ischemia-reperfusion, it loses its protective effect, and if an interval without preconditioning occurs about 2.5 to 3 days before ischemia-reperfusion interval, a second period of ischemic preconditioning can exert a protective effect 35 .It appears that persistent ischemic preconditioning leads to persistent stimulation of the cardiac opioid system and exerts a similar effect to persistent stimulation of these opioid receptors by agonists.Long-term and continuous stimulation of the opioid system may cause tolerance to its beneficial effects on the heart.Tolerance to the analgesic effect of opioids is a known effect 36 .
Contrary to our study results, which showed that chronic administration of methadone had no protective effect against ischemia-reperfusion injury, Peart et al. showed that chronic use of morphine for 5 days made the heart of C57/BL6 rats more resistant to IRI and that these protective effects were exerted through DOR and KOR.
In addition, the protection created in chronic conditions was about 20% higher than in acute administration, and even 48 h after withdrawal, the protective effect remained 37 .This difference can be due to the difference in the species of the studied animal, the duration of drug administration, the type of prescribed drug, and the type of receptor as methadone activates the mu receptor.
The opioid system is a fully integrated, coordinated, and complex system whose function is very precisely controlled, with many regulatory and compensatory mechanisms playing a role in its precise regulation 38 .This matter makes it highly adaptable.On the other hand, this feature makes it extremely vulnerable.For example, chronic exposure to morphine causes neurons to produce beta-endorphin 1-27, a weak morphine, instead of 1-31 beta-endorphin, which is a strong morphine.Therefore, in chronic use, not only are opioid receptors downregulated, but the system moves towards the production of weaker opioids 39 .In this study, as expected, ischemic preconditioning increased the phosphorylation of Akt (activation of the RISK pathway) and STAT3 (activation of the SAFE pathway).These two pathways are the main protective pathways activated by ischemic preconditioning and improve heart function after IRI.Methadone alone increased Akt phosphorylation but did not affect STAT3 phosphorylation.It appears that these changes in the RISK and SAFE pathways inhibit methadone's protective effect against IRI.In this regard, Goodman et al. showed in a study on male C57bl rats that in post-conditioning, the JAK-STAT signaling pathway is not able to create protective effects alone, without the activity of the PI3K-Akt pathway 40 .
Chronic use of methadone along with ischemic preconditioning caused a sharp increase in Akt phosphorylation, so the level of phosphorylation of this protein in the M-Pre-IR group was significantly higher than both the M-IR and Pre-IR groups.However, despite this sharp increase in activity in the RISK pathway, the protective effect of ischemic preconditioning was not observed.It has been shown that the continuous activation of Akt in the heart reduces cardiac resistance against myocardial infarction 41 .In addition, Akt hyperactivation inhibits the expression of antioxidant proteins through the inhibition of the forkhead family transcription factors 42 .Therefore, it appears that hyperactivation of Akt can be one of the reasons for the lack of protective effect of ischemic preconditioning.
Methadone reduces the size of the mitochondrial network, the number of mitochondrial objects, and increases the average area of mitochondrial objects.Also, methadone changes the shape of mitochondria.Thus, methadone can have a negative impact on mitochondrial structure, which may be linked to cell death 43 .Furthermore, in another study it was shown that methadone leads to necrotic-like cellular death by decreasing mitochondrial ATP synthesis, creating a bioenergetic crisis, and reducing the energy level of the cell 44 .Mitochondria play a crucial role in the cardioprotective mechanisms, particularly in the SAFE and RISK pathways 34 .Given the impact of methadone on mitochondrial function, it is plausible that chronic methadone administration can disrupt the cardioprotective mechanisms mediated by the SAFE and RISK pathways.
Research findings have demonstrated that Triiodothyronine and Gasterin can induce cardioprotection in isolated rat hearts by activating the RISK signaling pathway 45,46 .It would be intriguing to investigate whether activators of the RISK signaling pathway could have a cardioprotective effect in hearts with increased basal Akt activation due to chronic opioid receptor activation.This could potentially provide valuable insights into the mechanisms underlying cardioprotection in different physiological and pharmacological conditions.Further research in this area could help in understanding the complex interplay between different signaling pathways and their implications for heart health under various circumstances.
Ischemic conditioning, including preconditioning, postconditioning, and remote conditioning, have shown promising results in experimental studies for reducing ischemia-reperfusion injury in various organs, including the heart 5,47 .However, translating these strategies into clinical benefits for patients has been challenging 11 .One of the reasons for this challenge is the lack of understanding of the potential confounding factors that may impact the effectiveness of ischemic conditioning [48][49][50][51] .The present study highlights the potential role of chronic opioid receptor activation, specifically with methadone use, as a confounding factor that can inhibit the protective effects of preconditioning on ischemia-reperfusion injury in the heart.This suggests that chronic use of opioids, which is common in patients with chronic pain or opioid dependence, may interfere with the cardioprotective mechanisms of ischemic conditioning.In addition, our study helps to understand the mechanisms of ischemic preconditioning.Understanding its precise mechanisms has paved the way for the development of novel drugs and therapies that can mimic the effects of ischemic preconditioning and provide protection against ischemic injury, A limitation of our study is the absence of a control group receiving a single dose of methadone for comparison with those using methadone chronically.Another limitation of our study is that we investigated a moderate conditioning stimulus, which may not have been sufficient to induce maximum cardioprotective effects.The translation of ischemic conditioning for cardioprotection in clinical practice has been challenging 50,52 .To address this issue, certain factors should be considered in pre-clinical studies 53 .For example, it has been shown that there is a dose-response relationship between stimulus potency and induced cardioprotection 54 .Thus, future studies should consider including different dosing regimens of methadone and a stronger conditioning stimulus to better understand the optimal stimulus strength for cardioprotective interventions.

Conclusion
Chronic use of methadone eliminates the protective effects of preconditioning on IRI by changing the phosphorylation of Akt and STAT3 and possibly changing their signaling pathway.It appears that to observe the beneficial effects of cardiac preconditioning with opioids, special attention should be paid to the type of opioid and the duration of use.In addition, patients who use methadone and develop heart diseases may not be able to benefit from the beneficial effects of cardiac conditioning. https://doi.org/10.1038/s41598-024-65349-x https://doi.org/10.1038/s41598-024-65349-x